We are investigating the molecular and metabolic events involved in the initiation of several distinct adrenal glucocorticoid hormone actions in model systems that provide information about some of the more clinically important biological effects: suppression of inflammation, suppression of the immune response, cellular differentiation, and the suppression and killing of thymic lymphocytes and lymphoid cancer cells. The common thread is our working hypothesis that each of these actions is initiated through hormone-induced changes (increases or decreases) in one or more regulatory protein mediators. In our previous work we developed the evidence for such mediators through biochemical studies, devised novel means for their detection (ultra-high-resolution 2-dimensional gel electrophoresis), and discovered one or several rapidly-evolving inductions among the very-low-abundance rapidly-turning-over proteins (and their mRNAs) in each type of target cells; these are now recognized as candidates for mediators of the individual hormone actions. In the coming project period we are well into the second stage of this research, where the principal focus is on the purification, development of oligonucleotide and antibody probes, molecular cloning, characterization, sub-cellular location, and regulation of these proteins and their mRNAs. Since they are mostly of very low abundance some of this work requires the further development of innovative strategies for the cloning of the minor proteins resolved on 2-D gels. As the full-length cDNAs are obtained we are also entering a third stage, the determination of the functions for individual mediators. This will be accomplished by the construction and use of live virus and other expression vectors, and by other means. One mediator we have discovered, cloned, and are continuing to study is likely to have particular clinical relevance. It is a novel glucocorticoid-regulated inflammatory cyclooxygenase (gri-PGHS) that is dramatically increased in inflammation and remarkably suppressed by glucocorticoids. It is likely to play a central role in the pathogenesis in many if not all the inflammatory diseases that afflict mankind, and its suppression by glucocorticoids provides molecular basis for the understanding the most clinically useful of glucocorticoid actions, the suppression of inflammation.
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